European Journal of Immunology

Fibroblasts are traditionally considered structural cells that maintain tissue homeostasis and facilitate repair. However, accumulating evidence suggests they also participate in innate immunity, although their pattern recognition capabilities remain incompletely characterized. Here, we systematically assessed the innate immune responses of commercially available primary human dermal fibroblasts from a male and a female donor. Fibroblasts were stimulated with a panel of microbe-associated molecular patterns (MAMPs) targeting various pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NODs), Alpha kinase 1 (ALPK1) and STING. Innate immune activation was quantified by measuring the nuclear translocation of NF-{kappa}B via high content microscopy and cytokines and chemokines secretion by ELISA; baseline PRRs expression was determined by quantitative PCR. Only a restricted subset of agonists, specifically E. coli LPS (TLR4), Poly I:C (TLR3 / RIG-I) and unexpectedly ADP heptose (ALPK1) induced robust NF-{kappa}B activation and secretion of the chemokines IL-8 and MCP-1. Apart from IL-6 and RANTES, which were produced exclusively following Poly I:C stimulation, pro-inflammatory cytokines (IL-1{beta}, TNF, IFN-{beta}) and the anti-inflammatory cytokine IL-10 remained undetectable. Consistent with this limited reactivity, qPCR of PRRs revealed basal expression of TLR4 and ALPK1, whereas most other receptors were expressed at very low or undetectable levels. Notably, NOD1 was highly expressed although no cell activation was observed with several NOD1 agonists. Dose-response analysis revealed surprisingly high sensitivity to LPS. In conclusion, primary human dermal fibroblasts exhibit a highly selective but sensitive innate immune response, largely restricted to chemokine production upon PRR activation. This unexpected dissociation between chemokine and cytokine responses suggests that fibroblasts function as sentinel cells in early skin defense, capable of detecting key microbial patterns at low concentrations, to orchestrate local immune surveillance. Further investigation into interindividual variability and context-dependent activation is needed.

BackgroundImmunoglobulin E (IgE) plays a fundamental role in the pathogenesis of allergic disease, including asthma. The IgE-producing plasma cells (PCs) are thought to persist indefinitely, providing a sustained source of allergen-specific IgE. Although these cells can accumulate in the bone marrow (BM), after prolonged allergen exposure, their frequency remains remarkably low, and the mechanisms that regulate their migration are poorly understood. ObjectiveTo investigate the chemokine receptor profile and the migration potential of the human IgE-producing cells. MethodsTonsil B cells were stimulated with IL-4 and anti-CD40 to induce class switching to IgE and IgG1. The chemokine receptor profile of IgE+ and IgG1+ switched cells was determined using flow cytometry and migration towards relevant chemokines was quantified using transwell chemotaxis assays. Chemokine expression was also validated by re-analysis of a published single cell RNA sequencing (scRNAseq) dataset of PCs isolated from nasal polyps (NP) of patients with allergic fungal rhinosinusitis. ResultsIgE PCs exhibit significantly reduced expression of the BM-homing chemokine receptor CXCR4 and impaired migration towards its ligand, CXCL12. While IgE+ PCs can upregulate CCR10 and respond to its ligand, CCL28, this behaviour is similar to IgG1+ PCs. Strikingly, however, IgE PCs selectively upregulate CCR2 and migrate robustly towards its ligand CCL2. Re-analysis of NP scRNAseq data confirmed that IgE PCs express significantly higher levels of CCR2 compared with PCs of all other isotypes. ConclusionsThese findings identify CCR2 as a key regulator of IgE PC migration and provide insights into their homing preferences that may shape the nature of the IgE responses.

Immune phenotyping represents a pillar in diagnostics, characterization of new genetic defects, and understanding mechanisms of diseases. Cell population distribution often does not cover the intrinsic function changes that may contribute to disease. Outcome of signaling activation can be used as proxy for cell function. To overcome the limitation of sample availability and standardization of signaling assays, we developed a multiplex full spectrum cytometry phosphoflow assay allowing the study of 6 phospho-proteins representing BCR/TCR, MAPK, PI3K/Akt/mTOR and canonical NF-{kappa}B signaling pathways in 18 immune cell subpopulations. Maximal stimulation and temporal dynamics were studied in response to pan-stimuli, activating cells regardless of receptor, and targeted stimuli for T, B, and innate immune cells. We studied healthy individuals between 1-69 years and discovered subpopulations-specific responses. Furthermore, pediatric donors showed broad differences in B cell and T cell function compared to adults. Hence, we established a tool to assess multiple signaling pathways at once and provide age- and subpopulation-specific references for signaling outcome. SummaryMultiplex full spectrum flow cytometry-based phosphoflow assay across 18 immune cell subpopulations, 6 phospho-proteins in response to 6 stimuli at 4 time points in individuals aged 1-69 years, reveals distinct age- and subpopulation-associated signaling patterns in magnitude and dynamics of pathways activation.

Abstract Introduction: IL-15 is one of the most promising candidate cytokines in cancer immunotherapy due to its ability to promote the activity of different cytotoxic innate immune cell subsets such as NK, ILC1 and gammadelta T cells. During biosynthesis, IL-15 associates with IL-15alpha and is transported to the cell surface where IL-15Ralpha trans-presents IL-15 to target neighboring cells expressing the beta chain (IL-2Rbeta) and the common gamma chain. Our group previously showed that in autoimmune type 1 diabetes and early innate immune responses to infections trans-presentation by IL-15Ralpha is dispensable. Here we addressed the relative roles of IL-15 and trans-presented IL-15 in the control of established tumors and spontaneous tumor development. Methodology: Growth kinetics of tumor cell lines were monitored in WT, Il15-/- and Il15ra-/- mice. Spontaneous fibrosarcoma was induced with Methylcholanthrene (MCA) in WT, Il15-/- and Il15ra-/- mice. Cell lines were established from MCA-induced tumors to characterize their immunogenicity. Results: Growth of established tumor cell lines were comparable in the three genotypes. MCA-induced tumor incidence was reduced in Il15ra-/- mice when compared to WT and Il15-/- mice. In vitro, MCA tumor-derived cell lines expressed MHC-I and PD-L1 and had comparable proliferation rates. In vivo, MCA tumor-derived cell lines established from the 3 genotypes showed comparative growth in WT mice suggesting that IL-15 does not impact immunoediting. Nonetheless, NLRC5 expressing B16-F10 tumors were contained in WT and Il15ra-/- mice but not in Il15-/- mice. Conclusions: Taken together, these results show that in the absence of trans-presentation by IL-15Ralpha, IL-15 can better control spontaneous tumor development and that IL-15 signaling plays a minor role in immunosurveillance in this model. IL-15 signaling, independent of IL-15Ralpha has a significant role in the control of solid tumors.

Mesenchymal stromal cells exhibit potent immunomodulatory properties and are under active investigation for the treatment of immune-mediated disorders. However, their clinical translation is hindered by the lack of standardized potency assays. Here, we established a reproducible mixed lymphocyte reaction platform by systematically optimizing peripheral blood mononuclear cell donor composition, culture conditions, and co-culture ratios to define a robust activation window. Using this system, we compared bone marrow and adipose derived Mesenchymal stromal cells across independent donor batches. Both sources effectively suppressed T cell proliferation, with the adipocyte derived source consistently showing greater inhibitory activity, while a conserved lower threshold of suppression was observed across both sources. Mesenchymal stromal cells reduced early (CD25+) and late (CD25+HLA-DR+) T cell activation, with downregulation of these markers emerging as a sensitive correlate of functional potency. Notably, bone marrow derived mesenchymal stromal cells exerted stronger suppression on late-stage activation and preferentially suppressed CD8+ T cell expansion. Mechanistically, this immunosuppression was associated with modulation of the PD-1 pathway, characterized by decreased soluble PD-1, increased PD-L1, and induction of mesenchymal stromal cells derived PD-L2. PD-L2 levels inversely correlated with T cell proliferation, identifying a PD-1/PD-L2 regulatory axis linked to the cells potency. These findings define a standardized and mechanistically informed potency assay framework for assessing mesenchymal stromal cell immunomodulatory function.

Accurate identification and quantification of eosinophils is critical for the diagnosis and monitoring of eosinophil-associated disorders. While flow cytometry remains a powerful tool for leukocyte characterization, conventional instruments equipped with 405 nm or 488 nm side scatter (SSC) detectors offer limited resolution for eosinophil discrimination overlap in scatter with neutrophils. Using a spectral flow cytometer equipped with six distinct SSC detectors, we report a novel, label-free approach for eosinophil detection leveraging high 808 nm near-infrared SSC (IRSSC) uniquely observed in human eosinophils. This optical signature is independent of antibody labeling, activation fixation, or permeabilization, and shows strong concordance with conventional CD66b/CD16 gating strategies (R = 0.997). Notably, the high 808 nm SSC is absent in murine eosinophils, suggesting a species-specific structural feature such as in human eosinophils. These findings establish IRSSC as a robust, reagent-free biomarker for eosinophil detection, with broad implications for both clinical diagnostics and translational immunology.

Mounting evidence indicates that T cells can operate in an innate-like mode challenging the classical description of T cells as strictly adaptive immune effectors. T cells can engage innate pattern recognition receptors to mount rapid but antigen-nonspecific responses to infection or cellular stress. This study observed that CD8+ T cells, and to a lesser extent also CD4+ T cells, responded to viral proteins in the mouse lung quickly in an innate-like fashion. We employed intravital lung microscopy to visualize infiltration of CD8+ T cells into the lung following intratracheal instillation of the SARS-CoV-2 envelope (E)-protein. Here, we demonstrate acute recruitment of CD8+ from the pulmonary microcirculation into the lung as early as 4 and 24 hours after (E)-protein instillation. The acute infiltration of CD8+ T cells was not observed in Tlr2-/- mice. Immunohistochemistry analysis of mouse lungs revealed T cell accumulation in nodular inflammatory foci (NIF) of the lung at perivascular regions and around large airways. Stimulating spleen-derived CD8+ T cells from wild-type mice with (E)-protein ex vivo in combination with cytokines or TCR agonists significantly upregulated CD69 and activated secretion of interferon (IFN){gamma} which was not observed with CD8+ T cells isolated from Tlr2-/- mice. These findings indicate rapid bystander activation of CD8+ T cells by the SARS-CoV-2 envelope (E)-protein that depends on (E)-protein sensing by TLR2. This innate-like CD8+ T cell response to SARS-CoV-2 (E)-protein may offer novel opportunities for diagnostic and therapeutic development, warranting further investigation.

Tissue-resident peritoneal macrophages (pM[FE]) programmed by GATA6 are essential players regulating immunity and tissue balance in the peritoneal cavity. How GATA6 regulates global lipid metabolism is currently unknown. Addressing this, in myeloid-restricted Gata6-deficient (Gata6-KOmye) mice, significant changes to the pM[FE] lipidome were found. First, Bodipy staining and anti-Stokes Raman scattering (CARS) microscopy demonstrated significant intracellular lipid accumulation in lipid droplets in Gata6-KOmye. Untargeted and targeted lipidomics revealed this to result from increased levels of multiple sphingolipid (SL) molecular species, including sphingomyelins, ceramides, and glycosphingolipids, along with upregulation of the cysteinyl leukotriene (CysLTs) pathway at both lipidomic and transcriptional levels. Evidencing a functional role for the lipidomic phenotype, Gata6-KOmye showed significant eosinophil accumulation, associated with decreased apoptosis which was exclusively driven by CysLT signalling. In summary, GATA6 is demonstrated as a regulator of sphingolipid accumulation and CysLT generation in pM[FE], with secondary impacts on associated leukocytes through regulation of transcellular CysLT signaling.

Vaccine-induced immune responses are the result of an intricate interplay between different cell populations of the innate and adaptive immune system, which is so far only partly understood. In particular, the role of polymorphonuclear neutrophils (PMNs) has long been neglected. Here, we studied the effects of a whole inactivated virus influenza vaccine (WIV) in an in vitro system consisting of freshly isolated human PMNs alone or PMNs combined with autologous peripheral blood mononuclear cells (PBMCs). Isolated PMNs showed minimal responses to the vaccine with respect to apoptosis, gene expression, cytokine production, and reactive oxygen species production. However, in WIV-stimulated PMN/PBMC co-cultures, PMNs particularly enhanced monocyte dynamics, CD14-CD11c+ cell activation, effector T cell differentiation, and B cell antibody production. On the other hand, PMNs decreased T follicular helper cell frequencies. Without vaccine stimulation, PMN presence resulted in enhanced levels of baseline inflammatory cytokines in PMN/PBMC co-cultures. However, with vaccine stimulation, PMNs dampened the vaccine-induced cytokine secretion of PBMCs. These findings reveal PMNs as regulators of vaccine responses whose effects depend on crosstalk with other immune cells, balancing pro-inflammatory and adaptive immune activation. Author summaryPolymorphonuclear neutrophils (PMNs) are essential and predominant cells of the human innate immune system. Growing evidence implicates that PMNs are involved in vaccine-induced immune activation, but their exact role is so far poorly defined. In our study, human PMNs were tested alone to observe their response to whole inactivated virus influenza vaccine (WIV), or combined with autologous peripheral blood mononuclear cells (PBMCs) to investigate how their presence influences vaccine responses of various cell populations within PBMCs. Our results show that WIV had little direct effect on isolated PMNs. However, when PMNs were combined with other immune cells, PMNs acted as crucial regulators: they enhanced the activity of innate immune cells, regulated the responses to the vaccine of T and B cells, and helped control the overall level of inflammation. Our study forms the groundwork for a more comprehensive understanding of human immune cell interactions under vaccine stimulation.

T cell activation is associated with, and dependent upon, the upregulation of amino acid uptake from the extracellular environment. Uptake of the non-essential amino acid asparagine (Asn) is mediated via amino transporters such as Slc1a5 whilst Asn can be synthesized within cells that express asparagine synthetase (ASNS). Previous work demonstrated that initial activation of CD8+ T cells is perturbed in the absence of Asn, whereas effector cytotoxic T cells cells upregulate ASNS and lose their dependence on Asn uptake. By contrast, less is known of the role of Asn uptake and ASNS in CD4+ T cell responses. Here we demonstrate that CD4+ T cells are more reliant than CD8+ T cells on Asn uptake for initial activation, differentiation, metabolic reprogramming and regulation of autophagy. These phenotypes are associated with enhanced expression of ASNS in CD8+ as compared to CD4+ effector T cells.

Human immunoglobulin G4 (IgG4) shapes both protective and pathogenic immunity, influencing conditions ranging from allergy and autoimmunity to cancer. However, subclass-specific targeting of memory B cells remains challenging due to limited knowledge of their phenotypic and functional heterogeneity. Here, we show that IgG4+ memory B cells are overrepresented in a niche of FCER2+BAFFR+ memory B cells, can be classified in several subtypes, have a distinct transcription factor profile and are the only memory B cell subset to express sterile IGHE transcripts. While IgG4+ B cells display a unique transcriptional profile, only BAFFR, IL5Rb and the B cell receptor were upregulated on protein level on IgG4+ cells. IgG4+ B cells have normal repertoire diversity, but a distinct germline V-gene usage, suggesting IgG4 responses are driven by specific antigens. By labeling autoreactive B cells, we confirmed that MuSK myasthenia gravis (an archetypal IgG4-mediatedautoimmune disease) patients have a normal memory B cell profile and that autoreactive IgG4+ memory B cells are extremely rare. These results highlight the unique features of IgG4+ B cells, provide insight on potential subclass-specific therapeutic targets and point towards an antigen-driven IgG4 response within a largely non-autoreactive memory B cell pool.

T cell exhaustion within the tumor microenvironment drives CD8+ T cells into a dysfunctional state characterized by progressive loss of proliferative capacity and effector functions, thereby limiting anti-tumor immunity and therapeutic efficacy. To investigate the biochemical alterations associated with exhaustion, an in vitro model of CD8+ T cell exhaustion was established through chronic antigenic stimulation of murine OT-1 CD8+ T cells. Phenotypic, functional, metabolic, and transcriptional characterization confirmed the acquisition of an exhausted state. Single-cell Raman spectroscopy was subsequently employed to generate biochemical signatures of activated, and exhausted CD8+ T cells. Principal component analysis (PCA) of the Raman spectral data revealed distinct separation of these two cell subsets, reflecting underlying biochemical differences associated with their functional states. Differential Raman spectral features corresponding to nucleic acids, carbohydrates, proteins, and lipids contributed significantly to this segregation, reflecting altered metabolic and biosynthetic states during exhaustion progression. Classification of the spectral data using machine-learning algorithms enabled accurate segregation of activated and exhausted T cells. Collectively, this study demonstrates that single-cell Raman spectroscopy can distinguish exhausted CD8+ T cells in a label-free and non-destructive manner, highlighting its potential as a platform for immune profiling and monitoring T cell dysfunction.

Inflammatory skin diseases (ISDs) affect up to 25% of the global population. Yet, large-scale comparative single-cell RNA-sequencing (scRNA-seq) analyses between ISDs are still missing. Here, we integrated scRNA-seq datasets spanning 27 skin diseases from 50 studies, comprising over 2 million cells from 441 samples. Using the healthy skin cell atlas as reference, we could build a robust ISD atlas that enabled us to differentiate universal inflammatory signatures and disease-specific ones. This highlighted, for example, a shared gene program between keratinocytes in atopic dermatitis and parapsoriasis, not present in cutaneous T-cell lymphoma, confirms the plasticity of Th17 cells throughout ISDs, defines specific macrophage signatures in acne, and reveals a yet undescribed role of mural cells in ISDs. This demonstrates the power of the ISD atlas as a resource to resolve disease-specific immune mechanisms. The complete atlas is available through an interactive online portal at https://isd-atlas.derma.meduniwien.ac.at.

NK cells can form clonal populations demonstrating features of adaptive immunity, including long-term memory and at least partial antigenic specificity. Given the limited individual diversity of activating receptors, the nature of NK cell antigenic specificity remains elusive. To explore this riddle, we combined scRNA-Seq of ex vivo FACS-sorted NK cell subsets expressing specific KIR receptors, single-cell cloning and bulk RNA-Seq of in vitro cultured KIR2DS4 NK cell clones, transcriptomic profiling of antigen-stimulated NK cells, and in silico modeling of glycosylated KIR2DS4-peptide-HLA complexes. scRNA-Seq resolved 12-15 clusters per KIR subset with highly heterogeneous KIR, KLRC and NCR expression patterns, consistent with clonal lineages. Notably, those clusters demonstrated over 30 differentially expressed glycosyltransferase genes, potentially involved in post-translational modification of NK cell receptors. Single-cell-derived KIR2DS4 cultures exhibited clone-specific cytotoxic, chemokine and KIR receptor genes, and transcriptional differences in > 40 glycosyltransferases. In peptide culturing autologous assays, SARS-CoV-2 (KTFPPTEPK) and EBV (CRAKFKHLL) peptides elicited NK cell proliferation and distinct transcriptional programs linking cytotoxicity genes, KIR2DS4 and glycosyltransferases. Structural modeling revealed that N-linked glycosyl residues in specific regions of KIR2DS4 may alter its contacts and interaction with MHCI and the presented peptide. We conclude that KIR human NK cells comprise clonally imprinted populations with distinct glycosyltransferase expression profiles, and site-specific KIR2DS4 glycosylation may modulate interaction with peptide-MHCI complexes, suggesting a post-translational layer of clonal NK cell diversification as a clue to their antigenic specificity.

Behcets disease (BD) is a systemic inflammatory disease. It is considered as an autoinflammatory disease triggered by innate immunity rather than adaptive immunity. Human leukocyte antigen-B51 (HLA-B51) is the strongest genetic factor associated with BD. This study investigated how HLA class 1 molecules interact with innate immune cells and induce cytokine secretion. For this purpose, 293T cells transfected with a plasmid encoding HLA-B51 were cultured with natural killer (NK) cells obtained from healthy human donors. Within 24 h, the concentrations of interleukin-4 (IL-4), IL-8, and interferon-{gamma} (IFN-{gamma}) in the medium increased, indicating that NK cells secreted cytokines without undergoing cellular expansion for cytolysis. NK cells stimulated by nonself HLA-B51 produced IFN-{gamma} levels comparable to those produced by NK cells stimulated by self HLA-B51. NK cells carrying HLA-B51 were accurately recognized by overexpressing HLA-B51 on 293T cells. Moreover, ample intracellular IFN-{gamma} levels were detected in NK cells after stimulation with phorbol 12-myristate-13-acetate (PMA) plus ionomycin. KLRK1 (CD314)-positive cells mainly primarily accounted for IFN-{gamma}-producing cells, whereas KLRK1-negative cells did not. In contrast, both NCR1 (CD335)-positive and -negative cells contributed to IFN-{gamma} production. We next investigated whether HLA-B51 on the surface of 293T cells stimulates KLRK1 as a ligand causing IFN-{gamma} secretion. In masking experiments using anti-KLRK1 antibodies, NK cells with high levels of cell surface KLRK1 decreased the production of IFN-{gamma}. Conversely, human NK cell line KHYG1 cells also produced IFN-{gamma} in culture with 293T cells, but did not increase IFN-{gamma} through HLA-B51 stimulation. The mRNA expression of the signal adaptor protein HCST (DAP10) in KHYG1 cells was lower than that in NK cells, whereas the relative expression of IL-2RA in KHYG1 cells was higher than that in NK cells. These findings suggest that HLA-B51 can interact with KLRK1 on the NK cells inducing IFN-{gamma} secretion, whereas IL-2 signals outweigh HLA-51 stimulation in KHYG1 cells.

Neoantigens are cancer-specific antigens arising from genomic alterations. Single Amino Acid Variants (SAAVs) represent a primary class of these neoantigens. To evaluate the therapeutic potential of Neurofibromin 1 (NF1)-derived SAAVs - given that NF1 is frequently mutated in malignant brain tumors - we prioritized the 40 NF1 SAAVs determined to be HLA-A*02:01 binders using computational prediction coupled with experimental validation. To validate these predicted neoepitopes, we employed a two-tiered experimental approach in HLA-A*02:01 homozygous U87-MG cells. We first synthesized minigene constructs encoding the predicted neoepitopes, introduced them via lentiviral transfection and confirmed their expression by mass spectrometry (MS). Subsequently, we performed endogenous validation using pan-HLA immunoprecipitation mass spectrometry (IP-MS), confirming 4 (10 neoepitopes) of the 40 candidate SAAVs. We observed a discrepancy between in silico predictions and the observed sequences. Our endogenous peptidomics further revealed conserved peptide motifs and demonstrated that peptide selection for HLA presentation is transient. While our study substantiates the therapeutic feasibility of T-cell immunotherapies targeting NF1 mutations, these results underscore a limitation in current computational prediction. Our study highlights the necessity of experimental validation to refine neoantigen prioritization strategies.

Tumor-draining lymph nodes (tdLNs) are critical hubs of anti-tumor immunity but are also vulnerable to tumor-mediated immunosuppression. We analyzed T cell and B cell receptor (TCR/BCR) repertoires and transcriptomes from sentinel and non-sentinel lymph nodes of patients with melanoma from a historical pre-immune checkpoint inhibitor cohort (1994-2002) and an independent contemporary validation cohort (2022-2024). Melanoma-positive lymph nodes exhibited increased immune receptor clonality compared with tumor-free nodes. While average clonality showed no consistent association with outcome, the presence of extreme high-clonality outliers in individual lymph nodes was strongly associated with poor melanoma-specific survival. These outliers were characterized by a loss of lymphocyte-related genes and activation markers, an enrichment of melanocytic transcripts, and the suppression of immune signaling pathways, consistent with local immune dysfunction. Increased clonality was confined to lymph nodes and not observed in the peripheral blood. T cell responses to melanocyte differentiation antigens were infre-quently shared between lymph nodes and peripheral blood, highlighting immune compartmentalization.

Background & AimsCeliac disease (CeD) is an autoimmune disorder triggered by dietary gluten in genetically predisposed individuals, but environmental factors contributing to disease onset remain incompletely defined. Epidemiological studies implicate enterovirus infections as potential triggers. Here, we investigated the epithelial-intrinsic mechanisms by which coxsackievirus B1 (CVB1) infection may prime the intestine for CeD. MethodsHuman intestinal organoids were infected with CVB1 and analyzed using single-cell RNA sequencing to resolve lineage-specific responses. Interferon signaling and transglutaminase 2 (TG2) regulation were interrogated using type I interferon stimulation and pharmacologic JAK inhibition. ResultsCVB1 infection induced a robust epithelial antiviral program dominated by type I interferon signaling. This response was accompanied by marked upregulation of TG2 expression and enzymatic activity. Single-cell analysis localized TG2 induction to immature goblet-lineage cells, which exhibited strong interferon-stimulated gene activation and epithelial stress signatures. Mechanistically, IFN-/{beta} stimulation was sufficient to induce TG2 via JAK-STAT signaling, while JAK inhibition effectively suppressed both TG2 expression and activity. In parallel, CVB1 infection triggered coordinated mucin remodeling, including induction of MUC5AC, indicating interferon-linked epithelial reprogramming. Notably, these effects occurred independently of immune cell involvement, highlighting a cell-intrinsic pathway. ConclusionOur findings identify a direct epithelial mechanism linking enterovirus infection to TG2 activation via interferon-driven JAK-STAT signaling. This pathway provides a mechanistic bridge between viral infection and gluten peptide modification, a critical step in the onset of CeD. The reversibility of TG2 induction by JAK inhibition suggests a potential strategy to prevent virus-mediated priming of celiac disease.

The role of antigen-specific T cells responding to antigen is a topic of intense studies, and critical for mechanistic insight of diseases and development of therapeutic strategies. Methods for broad-scale detection of antigen-specific CD4 T cells are lacking, while such methods have demonstrated great value in exploring CD8 T cell response in health and disease. Furthermore, major histocompatibility complex II (MHCII) assays are technically challenging due to high HLA diversity, lower binding affinities, low frequencies of ex vivo antigen-specific CD4 T cells and several bottlenecks in production and peptide exchange of MHCII monomers. Here we use peptide-loaded MHCII (pMHCII) proteins multimerized on a barcode- and fluorophore-labelled dextran backbone to provide a method for the detection of peptide-specific CD4 T cells by using a large display of MHCII-associated peptides. We have established a protocol for MHCII production and peptide-exchange suitable for the generation of large libraries of peptide-MHCII complexes. We validate the use of such pMHCII complexes in the form of barcode-labelled MHCII multimers to detect antigen-specific CD4 T cells. We demonstrate that we can identify antigen specific CD4 T cells, using these DNA barcoded peptide-MHCII multimer. The multimer bound CD4 T cells were selected based on the fluorochrome signal, and the co-attached DNA barcodes were hereafter amplified and used to identify the peptide-MHCII response/binding. In cases where the peptide-specific CD4 T cells frequencies are very low, we expanded the cell population with peptide-pools and in the presence of IL2. The given CD4 T cell populations hereby reach a cell number allowing for the DNA-barcoded pMHCII multimers to detect responses otherwise missed out. Applying this technology, we utilized a panel of 150 peptides derived from human cytomegalo virus (CMV), Epstein barr virus (EBV), Influenza (Flu), SARS CoV 2 and SARS CoV1, Hepatitis B virus (HBV), and Hepatitis C virus (HCV) loaded onto HLA-DRB1*01:01 and DRB1*04:01 to screen peripheral blood mononuclear cells (PBMC). We assessed ex vivo responses in 16 participants with HCV infection, and successfully detected naturally occurring viral-specific CD4 T cells at frequencies as low as 0.004% of total CD4 T cells. The low-frequency responses, identified via the barcode screen, were rigorously validated using individual fluorophore-labelled tetramer staining after a peptide-driven expansion in 15 participants. Furthermore, we assessed the recognition of novel HCV epitopes in 11 additional participants. Through this, we identified a total of 12 distinct HCV epitopes, including 9 that have not been previously utilized in assays to detect CD4 T cells. Overall, this barcoded-multimer platform provides a powerful tool for the large-scale discovery of class II epitopes and the broad profiling of CD4 T cell specificities. This method will allow for in-depth analyses of immune interactions, provide a better understanding of the antigen-driven associations between CD4 and CD8 T cell responses, and help dissect the complexities of CD4 T cell protection in HCV infection.

Monocytes are critical regulators of allergic inflammation, whose functions are modified by IgE-driven processes. Monocytes are heterogeneous; comprised of multiple subsets which implies differential functions. In allergic inflammation, this heterogeneity is likely influenced by IgE-mediated effects. We sought to identify phenotypically distinct monocyte subsets related to allergic disease and then further delineate functional differences in cytokine release and antiviral responses. Using high dimensional spectral flow cytometry, we identified monocyte surface phenotypes directly related to surface levels of the high affinity IgE receptor (Fc{epsilon}RI) and surface-bound IgE. Fc{epsilon}RI+IgE+ monocytes, or FIMs, correlated with allergic disease and the level of atopy (i.e. serum IgE levels) of individual subjects. The FIM population also had differential surface expression of other molecules of monocyte maturation, which closely resembled a type 2 conventional dendritic cell (cDC2) phenotype. Functionally, FIMs had enhanced antiviral responses and IgE-driven IL-10 cytokine release. Finally, we showed that FIMs could be identified at higher levels in lung tissue from individuals with asthma. This study supports that atopic disease drives differential monocyte phenotypes, with the FIM population, specifically, as a more mature cell population closely related to dendritic cells with enhanced antiviral responses. The presence of monocytes in lung tissue during lethal asthma exacerbation further supports a role in regulating tissue inflammatory responses in allergic airway disease.